This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The activation and proliferation of T cells is negatively regulated by hematopoietic tyrosine phosphatase (HePTP), a 38 kDa class I non-receptor protein tyrosine phosphatase. HePTP inactivates the extracellular signal-regulated kinase 2 (Erk2), a mitogen-activated protein kinase (MAPK), by dephosphorylating Tyr185 in its activation loop. In addition to its C-terminal PTP catalytic domain, HePTP contains a short N-terminal kinase interaction motif (KIM), which is essential for Erk2 binding. The objectives of our research are to understand the molecular basis of substrate specificity of HePTP for Erk2 dephosphorylation by solving the crystal structures of the following: 1) the PTP catalytic domain of HePTP bound to a peptide corresponding to the dually phosphorylated activation loop of Erk2, and 2) full-length HePTP bound to full-length, dually phosphorylated Erk2 protein. In order to selectively populate these transient complexes, we have generated multiple constructs of HePTP in which their key catalytic residues have been mutated to serine or alanine. Those mutants with substantially reduced enzymatic activity but unaltered substrate affinity function as substrate-trapping mutants. We determined that mutating Cys270, the catalytic cysteine of HePTP, reduces phosphatase activity by several orders of magnitude than that of wild-type HePTP. Thus the Cys270 mutant may potentially serve as a substrate-trapping mutant. We obtained crystals from a mixture of the Erk2 peptide and the HePTP Cys270 mutant in conditions lacking both phosphate and phosphate analogs. Because WT-HePTP has previously been reported to crystallize only in conditions containing either phosphate or phosphate analogs, we believe that our crystals of the HePTP Cys270 mutant contain the Erk2 peptide bound at its active site.